POS0102 CALCIUM CONTAINING CRYSTALS ARE HIGHLY PREVALENT IN JOINT FLUID OF ADVANCED OSTEOARTHRITIS OF THE KNEE
Authors: T. Niessink, T. Jansen, M. Janssen, T. Welting, C. Otto
Journal: Annals of the Rheumatic Diseases (2023)

Background Calcium containing crystals in osteoarthritis (OA) are of interest in the field of rheumatology, as they potentially stimulate the NRLP-3 inflammasome and may become treatable with novel therapeutics. Previous studies using scanning electron microscopy and polarization microscopy (PLM) demonstrated a presence of calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals in early and advanced OA [1]. Calcium carbonate crystals have been found in cartilage specimens of advanced OA and have recently been shown to potentially occur early in development of cartilage calcification [2] Objectives In this study we attempt to identify calcium containing crystals in joint fluid of patients with late-stage OA using Raman spectroscopy. Methods Synovial fluid samples from advanced knee OA cases were collected from the Maastricht University Medical Center (MUMC+) experimental orthopaedics biobank. This biobank contains synovial fluid samples from patients undergoing total knee replacement surgery for advanced osteoarthritis. The population includes both males and females and all patients were above 18 years of age. Samples were frozen at -80°C. No preservatives were used. Maastricht University Medical Hospital (METC azM/UM) approved sample collection. Patients gave written informed consent before collection of their synovial fluid. Samples were analyzed with an integrated Raman polarized light microscope (H-iRPolM, Hybriscan Technologies B.V., Nijkerk, the Netherlands). For each sample, 20 µl of synovial fluid was pipetted on a microscope slide. Birefringent crystals were localized with PLM and then scanned with the integrated Raman spectrometer. For each particle, the Raman spectrum from 0-3600 cm -1 was measured for 1 s/pixel with a laser power of 10 mW and a 532 nm laser. Analysis time per particle was 1-2 minutes depending on particle size. Spectra were analysed by a trained analyst (TN, CO). Samples were considered positive for a certain type of crystal if at least one crystal with a distinctive spectrum could be identified. Results Calcium containing crystals were present in all (100%) of the 36 samples. 34 (94.4%) of the samples contained calcite (calcium carbonate) crystals. Dolomite (calcium magnesium carbonate) crystals were present in 10 (27.8%) samples, 9 (25.0%) samples contained both types of calcium carbonate crystals. CPP crystals were present in 33.3% of samples. All the CPP crystals were identified as triclinic CPP, amorphic or monoclinic CPP were not identified. Basic calcium phosphate crystals were present in 9 (25.0%) samples, all of which were hydroxyapatite. Calcium oxalate crystals were present in 7 (19.4%) samples, 3 of which were identified as calcium oxalate dihydrate (COD) and 4 of which were identified as calcium oxalate monohydrate. Most of the samples (80.6%) contained more than one type of calcium crystal, but CPP and BCP were never identified simultaneously in one sample. One patient had some MSU crystals. Raman spectra of identified particles are shown in figure 1. Figure 1. Raman spectra of identified calcium containing crystals. Conclusion Calcium containing crystals are common in synovial fluid samples from knee joints in patients with end-stage osteoarthritis. Raman spectroscopy is a novel technique for rheumatology which enables reliable detection and objective identification of crystals such as calcium carbonate. The observation of calcium carbonate (calcite) crystals in synovial fluids of OA patients is novel and a promising subject of further study. Pathways to presentation in synovial fluid and inflammatory properties should be investigated. References [1]Rosenthal, A.K., Crystals, inflammation, and osteoarthritis. Curr Opin Rheumatol, 2011. 23 (2): p. 170-3. [2]Casal-Beiroa, P., V. Balboa-Barreiro, N. Oreiro, et al., Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples. Diagnostics (Basel), 2021. 11 (3). Acknowledgements We want to thank ReumaNederland for funding this research. Disclosure of Interests Tom Niessink: None declared, Tim Jansen Shareholder of: Human Crystal Research B.V., Matthijs Janssen Shareholder of: Human Crystal Research B.V., Tim Welting: None declared, Cees Otto Shareholder of: Hybriscan Technologies B.V.